mir--183靶向抑制pdcd4促进食管鳞癌细胞增殖及侵袭

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1、南京医科大学硕士学位论文mechanismsinESCCdevelopment．Methods：1．UsingmicroarrayanalysistoscreenaberrantlyexpressedmiRNAsin3pairsofESCCtissuesandnormalcontrols，mil0183wasselectedastheobjectiveofthisstudy．2．TheexpressionofmiR-183in32pairsofESCCtissuesand30pairsofesophagealintraepithelialneop

2、lasiatissueswereexaminedbyTaqrmnqRT-PCRwhilepdcd4(mRNAandprotein)wasdetectedbySYBRGreenqRT-PCR,WesternblotandIHCassay．3．ThetargetgemsofmiR-183weresearchedwithbioinformaticstoolssuchasTargetScan／miRBaseandPictar,potentialtargetgenesexpressionwereassessedbyqRT-PCI乙Westernblotan

3、dLuciferasereporterassay．4．MiR-183mimics，miR-183inhibitors，si-pdcd4andtheircorrespondingnegativecontrolsweretransfectedintoESCCcells，CCK-8assayforcellprolifemtiort,colonyformationassayforcbnogenicgrowth,flowcytometryassayforcellapoptosis，transwellassayforcellmigrationandinvas

4、ion．5．DifferentialconcentrationsofP13眦TinhibitorLY294002(0rtM、10州、200M、40山VI)wereaddedintoESCCcellsandculturedforhours，miR-183levelWasmeasuredbyqRT-PCR,pdcd4，AKT,P-AKTexpressionsweredetectedbyWesternblotassay．Results：1．51up-regulatedand17down-regulatedmiRNAsinourmicmarray,wit

5、hmiR-183levelinESCCwas4．24timesthannormalcontrols．TheexpressionofmiR-183in32pairedofESCCtissuesWas3．98timesthannormalcontrols俨<0．0001)，while2．93timesinintraepithelialmophsiatissuesfP<0．005)．Theexpressionofpdcd4mRNAandproteininESCCtissueswereO．46and0．27timesthannormaltissuesfP

6、<0．05)．．5．南京医科大学硕士学位论文AccordingtotheIHCassay,thestainingofpdcd4proteininESCCtissueswasf0．178±0．021)，whichwassignificantlylowerthanthatofnormaltissues(0．267±0．031)伊<0．05)．2．Pdcd4wasonetargetgeneofmaR-183accordingtobioinforrmticsanalysis．Theexpressionofpdcd4proteinwasreduced(in

7、creased)whentmnsfectedwithmiR-183mimics(inhibitors)俨<0．05)whilepdcd4mRNAdidn’tchange(JP>0．05)．LuciferaseactivityWasdecreasedwhentransfectedpdcd4wt-3'-UTRvectorinmiR-183mimicscomparedtocontrol伊

8、ffectsofmiR-183mimicsonESCCcellsweredescribedusingCCK-8，colonyformat